Calibration of Trust in Automated Driving: A Matter of Initial Level of Trust and Automated Driving Style?

OBJECTIVE: Automated driving is becoming a reality, and such technology raises new concerns about human-machine interaction on road. This paper aims to investigate factors influencing trust calibration and evolution over time. BACKGROUND: Numerous studies showed trust was a determinant in automation use and misuse, particularly in the automated driving context. METHOD: Sixty-one drivers participated in an experiment aiming to better understand the influence of initial level of trust (Trustful vs. Distrustful) on drivers' behaviors and trust calibration during two sessions of simulated automated driving. The automated driving style was manipulated as positive (smooth) or negative (abrupt) to investigate human-machine early interactions. Trust was assessed over time through questionnaires. Drivers' visual behaviors and take-over performances during an unplanned take-over request were also investigated. RESULTS: Results showed an increase of trust over time, for both Trustful and Distrustful drivers regardless the automated driving style. Trust was also found to fluctuate over time depending on the specific events handled by the automated vehicle. Take-over performances were not influenced by the initial level of trust nor automated driving style. CONCLUSION: Trust in automated driving increases rapidly when drivers' experience such a system. Initial level of trust seems to be crucial in further trust calibration and modulate the effect of automation performance. Long-term trust evolutions suggest that experience modify drivers' mental model about automated driving systems. APPLICATION: In the automated driving context, trust calibration is a decisive question to guide such systems' proper utilization, and road safety.

How drowsiness and distraction can interfere with take-over performance: A systematic and meta-analysis review.

Drowsiness and distraction are major factors of road crashes and responsible of>35% of road fatalities. Automated driving could solve or minimize their impact, yet it is also in itself a way to promote them. Previous literature reviews and meta-analysis regarding take-overs during automated driving primarily focused on distraction rather than drowsiness. We thus present a systematic and meta-analysis literature review focused on the effect of distraction and drowsiness on take-over performance. From an initial selection of 1896 articles from databases, we obtained by applying systematic review methodology a total of 58 articles with 42 articles dedicated to distraction and 17 articles related to drowsiness. According to our analysis, we demonstrated that distraction and drowsiness increased the take-over request reaction time (TOR-RT), which could also lead to a reduction of the quality of take-overs. In addition, this longer reaction time was even more important in the case of handheld non-driving related tasks, which is important to consider as phone use is among the most frequent tasks done during automated driving. On a more methodological aspect, we also demonstrated that take-over time budget had a significant effect on TOR-RT. However, it is difficult to estimate to what extend distraction and drowsiness could impact the take-over quality, even if several elements supported safety issues. We underpinned several limits of the current methodologies applied in the study of distraction and drowsiness such as (i) the lack of additional measures related to the take-over quality (e.g., accelerations, collision rate), (ii) the many different methodologies applied to the determination of the TOR-RT (e.g., deactivation by the steering wheel, pedals, button), (iii) the high frequency of take-over requests which can lead to habituation effects, (iv) the lack of control conditions, (v) the fact that the level of drowsiness was relatively low in most studies. We thus highlighted recommendations for a better estimation of the effect of distraction and drowsiness on take-over performance. Further studies should adopt more standardized measures of TOR-RT and additional take-over quality measures, try minimizing the number of take-over requests, and carefully consider the time budget available for the use case since it influences the TOR-RT. Regarding distraction, researchers should consider the impact of tasks requiring handholding items. Concerning drowsiness, further protocols should consider the non-linearity of drowsiness and presence of micro sleeps and favor take-over requests based on drowsiness level protocols rather than on fixed duration protocols.

New insights into hydroxyectoine synthesis and its transcriptional regulation in the broad-salt growing halophilic bacterium Chromohalobacter salexigens.

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C.␣salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyectoine production.

The impact of false warnings on partial and full lane departure warnings effectiveness and acceptance in car driving.

In the past, lane departure warnings (LDWs) were demonstrated to improve driving behaviours during lane departures but little is known about the effects of unreliable warnings. This experiment focused on the influence of false warnings alone or in combination with missed warnings and warning onset on assistance effectiveness and acceptance. Two assistance unreliability levels (33 and 17%) and two warning onsets (partial and full lane departure) were manipulated in order to investigate interaction. Results showed that assistance, regardless unreliability levels and warning onsets, improved driving behaviours during lane departure episodes and outside of these episodes by favouring better lane-keeping performances. Full lane departure and highly unreliable warnings, however, reduced assistance efficiency. Drivers' assistance acceptance was better for the most reliable warnings and for the subsequent warnings. The data indicate that imperfect LDWs (false warnings or false and missed warnings) further improve driving behaviours compared to no assistance. Practitioner Summary: This study revealed that imperfect lane departure warnings are able to significantly improve driving performances and that warning onset is a key element for assistance effectiveness and acceptance. The conclusion may be of particular interest for lane departure warning designers.

Oseltamivir treatment for influenza in hospitalized children without underlying diseases.

AIM: To determine whether the treatment with oseltamivir improves the outcome of children with confirmed influenza infection and no other underlying disease. METHODS: Multicentric, retrospective study performed in 10 hospitals of Madrid between September 2010 and June 2012. All children admitted to the hospitals with confirmed influenza infections were eligible. Children with risk factors for serious disease and nosocomial influenza infections were excluded. Asthma was not considered an exclusion factor. The study compared patients treated and untreated with oseltamivir. Fever duration, oxygen support, antibiotics administration, length of hospital stay, intensive care admission and bacterial complications were analyzed. To compare variables, χ(2) test, Fisher exact test, ANOVA or Mann-Whitney U test were used. RESULTS: Two hundred eighty-seven children were included and 93 of them were treated with oseltamivir (32%). There were no significant differences between treated and untreated patients in days of fever after admission (1.7 ± 2; 2.1 ± 2.9, P > 0.05), length of stay (5.2 ± 3.6; 5.5 ± 3.4, P > 0.05), days of hypoxia (1.6 ± 2.3; 2.1 ± 2.9, P > 0.05), diagnosis of bacterial pneumonia (10%; 17%, P > 0.05), intensive care admission (6.5%; 1.5%,P > 0.05) or antibiotic prescription (44%; 51%, P > 0.05). There were no differences when the population was stratified by age (below or over 1 year) or by the presence or absence of asthma. CONCLUSIONS: There were no proven benefits of treatment with oseltamivir in hospitalized pediatric patients without the underlying diseases or risk factors for developing a serious illness, including those with asthma.

Effectiveness of a Forward Collision Warning System in simple and in dual task from an electrophysiological perspective.

Forward Collision Warning Systems (FCWS) are expected to assist drivers; however, it is not completely clear whether these systems are of benefit to distracted drivers as much as they are to undistracted drivers. This study aims at investigating further the analysis of the effectiveness of a surrogate FCWS according to the attentional state of participants. In this experiment electrophysiological and behavioural data were recording while participants were required to drive in a simple car simulator and to react to the braking of the lead vehicle which could be announced by a warning system. The effectiveness of this warning system was evaluated when drivers were distracted or not by a secondary cognitive task. In a previous study, the warning signal was not completely effective likely due to the presence of another predictor of the forthcoming braking which competes with the warning. By eliminating this secondary predictor in the present study, the results confirmed the negative effect of the secondary task and revealed the expected effectiveness of the warning system at behavioural and electrophysiological levels.

Impact of totally and partially predictive alert in distracted and undistracted subjects: An event related potential study.

Rear-end collisions represent about 30% of all car crashes and generate a significant economic cost for society. Driver inattention has been identified as the most important contributing factor in rear-end collisions. One possible countermeasure is the use of systems that warn drivers of potential collisions. Nevertheless, because of technical constraints, the conception of perfect warning systems is difficult to achieve and technical literature shows that these kinds of systems can be prone to false alerts or misses. The main objective of this study is to assess the impact of such a warning system on the processing of a relevant driving visual cue while taking into account the reliability of the system and the attentional state of the participants. For this, we designed a laboratory experiment during which we recorded behavioral data and brain activity (event related potential, ERP) following the detection of a visual target. Three warning conditions were designed: (1) no alert was presented before the visual target; (2) an auditory alert was presented before each target; (3) an alert was presented before the target in 70% of the trials (15% only had the alert without the target, and 15% only had the target without the alert). In addition, participants had to perform this visual detection task either alone (simple task) or with a concurrent problem-solving task (dual task). Behavioral and electrophysiological data contribute to revealing (1) that there is a behavioral gain induced by the alert and (2) that this gain is at least linked with a time-saving aspect at both the sensory and cognitive stages of neural information processing. Nevertheless, this impact depends on the attentional states of the participant and on the reliability of the alert.

Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli.

BACKGROUND: The compatible solute trehalose is involved in the osmostress response of Rhizobium etli, the microsymbiont of Phaseolus vulgaris. In this work, we reconstructed trehalose metabolism in R. etli, and investigated its role in cellular adaptation and survival to heat and desiccation stress under free living conditions. RESULTS: Besides trehalose as major compatible solute, R. etli CE3 also accumulated glutamate and, if present in the medium, mannitol. Putative genes for trehalose synthesis (otsAB/treS/treZY), uptake (aglEFGK/thuEFGK) and degradation (thuAB/treC) were scattered among the chromosome and plasmids p42a, p42c, p42e, and p42f, and in some instances found redundant. Two copies of the otsA gene, encoding trehalose-6-P-synthase, were located in the chromosome (otsAch) and plasmid p42a (otsAa), and the latter seemed to be acquired by horizontal transfer. High temperature alone did not influence growth of R. etli, but a combination of high temperature and osmotic stress was more deleterious for growth than osmotic stress alone. Although high temperature induced some trehalose synthesis by R. etli, trehalose biosynthesis was mainly triggered by osmotic stress. However, an otsAch mutant, unable to synthesize trehalose in minimal medium, showed impaired growth at high temperature, suggesting that trehalose plays a role in thermoprotection of R. etli. Desiccation tolerance by R. etli wild type cells was dependent of high trehalose production by osmotic pre-conditioned cells. Cells of the mutant strain otsAch showed ca. 3-fold lower survival levels than the wild type strain after drying, and a null viability after 4 days storage. CONCLUSIONS: Our findings suggest a beneficial effect of osmotic stress in R. etli tolerance to desiccation, and an important role of trehalose on the response of R. etli to high temperature and desiccation stress.

An electrophysiological study of the impact of a Forward Collision Warning System in a simulator driving task.

Driver distraction has been identified as the most important contributing factor in rear-end collisions. In this context, Forward Collision Warning Systems (FCWS) have been developed specifically to warn drivers of potential rear-end collisions. The main objective of this work is to evaluate the impact of a surrogate FCWS and of its reliability according to the driver's attentional state by recording both behavioral and electrophysiological data. Participants drove following a lead motorcycle in a simplified simulator with or without a warning system which gave forewarning of the preceding vehicle braking. Participants had to perform this driving task either alone (simple task) or simultaneously with a secondary cognitive task (dual task). Behavioral and electrophysiological data contributed to revealing a positive effect of the warning system. Participants were faster in detecting the brake light when the system was perfect or imperfect, and the time and attentional resources allocation required for processing the target at higher cognitive level were reduced when the system was completely reliable. When both tasks were performed simultaneously, warning effectiveness was considerably affected at both performance and neural levels; however, the analysis of the brain activity revealed fewer differences between distracted and undistracted drivers when using the warning system. These results show that electrophysiological data could be a valuable tool to complement behavioral data and to have a better understanding of how these systems impact the driver.

Role of trehalose in salinity and temperature tolerance in the model halophilic bacterium Chromohalobacter salexigens.

The disaccharide trehalose is considered as a universal stress molecule, protecting cells and biomolecules from injuries imposed by high osmolarity, heat, oxidation, desiccation and freezing. Chromohalobacter salexigens is a halophilic and extremely halotolerant γ-proteobacterium of the family Halomonadaceae. In this work, we have investigated the role of trehalose as a protectant against salinity, temperature and desiccation in C. salexigens. A mutant deficient in the trehalose-6-phosphate synthase gene (otsA::Ω) was not affected in its salt or heat tolerance, but double mutants ectoine- and trehalose-deficient, or hydroxyectoine-reduced and trehalose-deficient, displayed an osmo- and thermosensitive phenotype, respectively. This suggests a role of trehalose as a secondary solute involved in osmo- (at least at low salinity) and thermoprotection of C. salexigens. Interestingly, trehalose synthesis was osmoregulated at the transcriptional level, and thermoregulated at the post-transcriptional level, suggesting that C. salexigens cells need to be pre-conditioned by osmotic stress, in order to be able to quickly synthesize trehalose in response to heat stress. C. salexigens was more sensitive to desiccation than E. coli and desiccation tolerance was slightly improved when cells were grown at high temperature. Under these conditions, single mutants affected in the synthesis of trehalose or hydroxyectoine were more sensitive to desiccation than the wild-type strain. However, given the low survival rates of the wild type, the involvement of trehalose and hydroxyectoine in C. salexigens response to desiccation could not be firmly established.

A driving-emulation task to study the integration of goals with obligatory and prohibitory traffic signs.

This research aims to analyse how drivers integrate the information provided by traffic signs with their general goals (i.e. where they want to go). Some previous studies have evaluated the comparative advantages of obligatory and prohibitory traffic signs using a judgement task. In this work, a new experimental task with greater similarity to driving situations is proposed. Participants imagine they are driving a vehicle and must make right or left turn manoeuvres according to a previously indicated objective and the information from obligatory and prohibitory traffic signs. Eighty-two participants took part in two different experiments. According to the results, an obligatory traffic sign is associated with faster and more accurate responses only when the participant's initial objective is allowed. When the initial objective was not allowed, an advantage in accuracy was observed with prohibitory traffic signs and there was no significant difference in reaction time between the two types of sign. These results suggest that having an obligatory traffic sign may facilitate a correct response when the driver's goal is effectively allowed, whereas a prohibitory traffic sign could be more effective in preventing error when the driver has a not-allowed goal in mind. However, processing a prohibitory sign requires an extra inference (i.e. deciding which is the allowed manoeuvre), and thus the potential advantage in reaction time of the prohibitory sign may disappear. A second experiment showed that the results could not be explained by a potential congruency effect between the location (left or right) of the road signs and the position of the key or the hand used to respond (such as the Simon effect or the spatial Stroop effect). Also, an increase in the difficulty of the task (using an incongruent hand to respond) affected performance more strongly in experimental conditions that required making inferences. This made the advantage of the obligatory sign over the prohibitory sign in this condition more noteworthy. The evidence gathered in the current study could be of particular interest in some applied research areas, such as the assessment of road traffic signalling strategies or the ergonomic design of GPS navigation systems.

An extended suite of genetic tools for use in bacteria of the Halomonadaceae: an overview.

Halophilic gammaproteobacteria of the family Halomonadaceae (including the genera Aidingimonas, Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola, and Zymobacter) have current and promising applications in biotechnology mainly as a source of compatible solutes (powerful stabilizers of biomolecules and cells, with exciting potentialities in biomedicine), salt-tolerant enzymes, biosurfactants, and extracellular polysaccharides, among other products. In addition, they display a number of advantages to be used as cell factories, alternative to conventional prokaryotic hosts like Escherichia coli or Bacillus, for the production of recombinant proteins: (1) their high salt tolerance decreases to a minimum the necessity for aseptic conditions, resulting in cost-reducing conditions, (2) they are very easy to grow and maintain in the laboratory, and their nutritional requirements are simple, and (3) the majority can use a large range of compounds as a sole carbon and energy source. In the last 15 years, the efforts of our group and others have made possible the genetic manipulation of this bacterial group. In this review, the most relevant and recent tools for their genetic manipulation are described, with emphasis on nucleic acid isolation procedures, cloning and expression vectors, genetic exchange mechanisms, mutagenesis approaches, reporter genes, and genetic expression analyses. Complementary sections describing the influence of salinity on the susceptibility of these bacteria to antimicrobials, as well as the growth media most routinely used and culture conditions, for these microorganisms, are also included.

Pandemic H1N1 influenza-associated hospitalizations in children in Madrid, Spain.

OBJECTIVE: To describe the epidemiological and clinical characteristics of children hospitalized with 2009 pandemic influenza (pH1N1) in Madrid, Spain. PATIENTS/METHODS: We included patients less than 14 years of age admitted to one of 18 hospitals in Madrid, Spain, between May 1 and November 30, 2009 and diagnosed with pH1N1 by polymerase chain reaction. A retrospective chart review was conducted and data were compared by age, presence of high-risk medical conditions, and pediatric intensive care unit (PICU) admission. RESULTS: A total of 517 pH1N1 cases were included for final analysis. One hundred and forty-two patients (27·5%) had predisposing underlying illnesses, with immunosuppression (36 children, 7%) and moderate persistent asthma (34, 6·6%) being the most common ones. Patients with underlying medical conditions had longer hospital stays [median 5, interquartile range (IQR) 3-8 days, versus median 4, IQR 3-6, P < 0·001] and required intensive care (20·4% versus 5·9%, P < 0·001) and mechanical ventilation more frequently than previously healthy children. Globally, intensive care was required for 51 patients (10%) and invasive mechanical ventilation for 12 (2%). Pediatric intensive care unit admission was significantly associated with abnormal initial chest X-ray [Odds Ratio (OR) 3·5, 95% confidence interval (CI) 1·5-8·5], underlying neurological condition (OR 3·1, CI 1·2-7·5) and immunosuppression (OR 2·9, 1·2-6·8). Five patients (0·9%) died; two with severe neurological disease, two with leukemia, and one with a malignant solid tumor. CONCLUSIONS: Children with underlying medical conditions experienced more severe pH1N1 disease. Risk factors for admission to the PICU included underlying neurological conditions, immunosuppression and abnormal initial chest X-ray.

Laboratory-based, 2-year surveillance of pediatric parapneumonic pneumococcal empyema following heptavalent pneumococcal conjugate vaccine universal vaccination in Madrid.

BACKGROUND: In October 2006, the heptavalent pneumococcal conjugate vaccine was included in the Madrid vaccination calendar, warranting serotype (St) surveillances in pneumococcal pediatric parapneumonic empyema (PPE). METHODS: A prospective 2-year (May 2007-April 2009) laboratory-confirmed PPE surveillance was performed in 22 hospitals. All isolates (for serotyping) and culture-negative pleural fluids were sent to the reference laboratory for polymerase chain reaction (PCR) analysis. RESULTS: We identified 138 PPEs. Pneumococcal etiology was confirmed in 100 cases: 38 by culture, 62 by PCR. Mean age was 44.64 ± 26.64 months; 51.0% were male. Similar pneumococcal PPE distribution was found by age: 21% to 28% in <24, ≥24-<36, ≥36-<60, and ≥60 months. PPE-associated Sts were St 1 (38%), St 5 (15%), St 19A (11%), St 7F (9%), St 3 (8%), and others (19%). St 1 was the most common in >36 months, with similar rates to St 19A in <24 months (≈30%). In ≥24-≤36 months, St 3 (21.7%), St 1 and St 5 (17.4% each) were the most frequent. No differences in demographic data, vaccination status, length of hospitalization, and outcome were found between culture-negative (PCR positive) and culture-positive PPE patients, with significantly higher percentages of St 1 and St 5 in culture-positive PPEs. Total rates of St 1 (38%), St 5 (15%), and St 7F (9%) would have been over-represented considering only positive-culture PPEs (n = 38), by increasing to 52.6% (St 1), 23.7% (St 5), and 10.5% (St 7F). The 13-valent pneumococcal conjugate vaccine would cover 84.0% of Sts causing PPEs. CONCLUSIONS: PCR is essential for determining the specific etiology of PPE.

Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens.

BACKGROUND: Osmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions. RESULTS: A C. salexigens Tn1732-induced mutant (CHR95) showed a delayed growth with glucose at low and optimal salinities, could not grow at high salinity, and was able to use ectoines as carbon sources at low salinity. CHR95 was affected in the transport and/or metabolism of glucose, and showed a deregulated ectoine uptake at any salinity, but it was not affected in ectoine metabolism. Transposon insertion in CHR95 caused deletion of three genes, Csal0865-Csal0867: acs, encoding an acetyl-CoA synthase, mntR, encoding a transcriptional regulator of the DtxR/MntR family, and eupR, encoding a putative two-component response regulator with a LuxR_C-like DNA-binding helix-turn-helix domain. A single mntR mutant was sensitive to manganese, suggesting that mntR encodes a manganese-dependent transcriptional regulator. Deletion of eupR led to salt-sensitivity and enabled the mutant strain to use ectoines as carbon source at low salinity. Domain analysis included EupR as a member of the NarL/FixJ family of two component response regulators. Finally, the protein encoded by Csal869, located three genes downstream of eupR was suggested to be the cognate histidine kinase of EupR. This protein was predicted to be a hybrid histidine kinase with one transmembrane and one cytoplasmic sensor domain. CONCLUSIONS: This work represents the first example of the involvement of a two-component response regulator in the osmoadaptation of a true halophilic bacterium. Our results pave the way to the elucidation of the signal transduction pathway involved in the control of ectoine transport in C. salexigens.

Ectoines in cell stress protection: uses and biotechnological production.

Microorganisms produce and accumulate compatible solutes aiming at protecting themselves from environmental stresses. Among them, the wide spread in nature ectoines are receiving increasing attention by the scientific community because of their multiple applications. In fact, increasing commercial demand has led to a multiplication of efforts in order to improve processes for their production. In this review, the importance of current and potential applications of ectoines as protecting agents for macromolecules, cells and tissues, together with their potential as therapeutic agents for certain diseases are analyzed and current theories for the understanding of the molecular basis of their biological activity are discussed. The genetic, biochemical and environmental determinants of ectoines biosynthesis by natural and engineered producers are described. The major limitations of current bioprocesses used for ectoines production are discussed, with emphasis on the different microorganisms, environments, molecular engineering and fermentation strategies used to optimize the production and recovery of ectoines. The combined application of both bioprocess and metabolic engineering strategies, allowing a deeper understanding of the main factors controlling the production process is also stated. Finally, this review aims to summarize and update the state of the art in ectoines uses and applications and industrial scale production using bacteria, emphasizing the importance of reactor design and operation strategies, together with the metabolic engineering aspects and the need for feedback between wet and in silico work to optimize bioproduction.

Biosynthesis of compatible solutes in rhizobial strains isolated from Phaseolus vulgaris nodules in Tunisian fields.

BACKGROUND: Associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899T. RESULTS: The most NaCl-tolerant strain was A. tumefaciens 10c2, followed (in decreasing order) by R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3 and R. gallicum bv. phaseoli 8a3. 13C- and 1H-NMR analyses showed that all Rhizobium strains synthesized trehalose whereas A. tumefaciens 10c2 synthesized mannosucrose. Glutamate synthesis was also observed in R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3 and A. tumefaciens 10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in R. tropici CIAT 899 and of mannosucrose in A. tumefaciens 10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the otsA gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. In vivo 13C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the beta-1,2-cyclic glucan from R. tropici CIAT 899 was co-extracted with the cytoplasmic compatible solutes and its chemical structure was determined. CONCLUSIONS: The soil bacteria analyzed in this work accumulated mainly disaccharides in response to NaCl stress. We could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance, suggesting that additional osmoadaptive mechanism should be operating in the most NaCl-tolerant strain R. tropici CIAT 899.

Unravelling the adaptation responses to osmotic and temperature stress in Chromohalobacter salexigens, a bacterium with broad salinity tolerance.

Chromohalobacter salexigens, a Gammaproteobacterium belonging to the family Halomonadaceae, shows a broad salinity range for growth. Osmoprotection is achieved by the accumulation of compatible solutes either by transport (betaine, choline) or synthesis (mainly ectoine and hydroxyectoine). Ectoines can play additional roles as nutrients and, in the case of hydroxyectoine, in thermotolerance. A supplementary solute, trehalose, not present in cells grown at 37 degrees C, is accumulated at higher temperatures, suggesting its involvement in the response to heat stress. Trehalose is also accumulated at 37 degrees C in ectoine-deficient mutants, indicating that ectoines suppress trehalose synthesis in the wild-type strain. The genes for ectoine (ectABC) and hydroxyectoine (ectD, ectE) production are arranged in three different clusters within the C. salexigens chromosome. In order to cope with changing environment, C. salexigens regulates its cytoplasmic pool of ectoines by a number of mechanisms that we have started to elucidate. This is a highly complex process because (i) hydroxyectoine can be synthesized by other enzymes different to EctD (ii) ectoines can be catabolized to serve as nutrients, (iii) the involvement of several transcriptional regulators (sigmaS, sigma32, Fur, EctR) and hence different signal transduction pathways, and (iv) the existence of post-trancriptional control mechanisms. In this review we summarize our present knowledge on the physiology and genetics of the processes allowing C. salexigens to cope with osmotic stress and high temperature, with emphasis on the transcriptional regulation.

The ectD gene, which is involved in the synthesis of the compatible solute hydroxyectoine, is essential for thermoprotection of the halophilic bacterium Chromohalobacter salexigens.

The halophilic bacterium Chromohalobacter salexigens synthesizes and accumulates compatible solutes in response to salt and temperature stress. (13)C-nuclear magnetic resonance analysis of cells grown in minimal medium at the limiting temperature of 45 degrees C revealed the presence of hydroxyectoine, ectoine, glutamate, trehalose (not present in cells grown at 37 degrees C), and the ectoine precursor, Ngamma-acetyldiaminobutyric acid. High-performance liquid chromatography analyses showed that the levels of ectoine and hydroxyectoine were maximal during the stationary phase of growth. Accumulation of hydroxyectoine was up-regulated by salinity and temperature, whereas accumulation of ectoine was up-regulated by salinity and down-regulated by temperature. The ectD gene, which is involved in the conversion of ectoine to hydroxyectoine, was isolated as part of a DNA region that also contains a gene whose product belongs to the AraC-XylS family of transcriptional activators. Orthologs of ectD were found within the sequenced genomes of members of the proteobacteria, firmicutes, and actinobacteria, and their products were grouped into the ectoine hydroxylase subfamily, which was shown to belong to the superfamily of Fe(II)- and 2-oxoglutarate-dependent oxygenases. Analysis of the ectoine and hydroxyectoine contents of an ectABC ectD mutant strain fed with 1 mM ectoine or hydroxyectoine demonstrated that ectD is required for the main ectoine hydroxylase activity in C. salexigens. Although in minimal medium at 37 degrees C the wild-type strain grew with 0.5 to 3.0 M NaCl, with optimal growth at 1.5 M NaCl, at 45 degrees C it could not cope with the lowest (0.75 M NaCl) or the highest (3.0 M NaCl) salinity, and it grew optimally at 2.5 M NaCl. The ectD mutation caused a growth defect at 45 degrees C in minimal medium with 1.5 to 2.5 M NaCl, but it did not affect growth at 37 degrees C at any salinity tested. With 2.5 M NaCl, the ectD mutant synthesized 38% (at 37 degrees C) and 15% (at 45 degrees C) of the hydroxyectoine produced by the wild-type strain. All of these data reveal that hydroxyectoine synthesis mediated by the ectD gene is thermoregulated and essential for thermoprotection of C. salexigens.